Cancer treatment targeted to tumor adaptive responses to protein synthesis stress

ABSTRACT

In cancers such as prostate cancer, the combination of PTEN loss and activation of Myc activates an adaptive stress response that enables tumor cells to escape the stress of massively upregulated protein synthesis. This pro-survival response is mediated by the PERK-phosphorylated eIF2α axis of the UPR adaptive response. Agents that disrupt PERK-eIF2α pathways disrupt the adaptive response and lead to cancer cell death from uncontrolled growth. For example, ISRIB and derivatives may be employed as therapeutic agents to disrupt PERK-mediated adaptive mechanisms. Additionally PTEN loss and activation of Myc provides a diagnostic marker that enables better prognosis and the selection of amenable treatments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 62/664,144 entitled “Cancer Treatment Targeted toTumor Adaptive Responses to Protein Synthesis Stress,” filed Apr. 28,2018, the contents which are hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number R01CA154916 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Multiple oncogenes are known to be drivers of cancer. For example,phosphatase and tensin homolog, “PTEN,” as known in the art, is a tumorsuppressor gene which is commonly lost by mutation in tumor cells. Forexample, over 70% of prostate cancer patients have mutations in PTEN.PTEN loss is generally associated with dysregulation and upregulation ofprotein synthesis and other processes central to cancer.

Similarly, the Myc oncogenic transcription factor is a key regulator ofcell growth and proliferation. Activation of Myc, by mutations whichcause its constitutive expression, are known to cause the dysregulationof numerous downstream genes and is thought to play a role in manycancers. Myc activation has also been associated with upregulation ofprotein synthesis rates. Various Myc mutations which cause activationare known.

The combination of Myc activation and loss of PTEN is known in the artto exert a substantial impact on the progression of certain cancers. Forexample, in prostate cancer, this combination has been shown to resultin genetic instability and may lead to lethal metastatic prostatecancer. The combined loss of PTEN and Myc activation is also implicatedin other cancers, such as T-cell acute lymphoblastic leukemia.

In prostate and other cancers, uncontrolled cell growth and division issustained by stimulating the production of molecular building blocks,such as proteins and outputs of anabolic metabolism. One of the earlyconsequences of many cancers is a major remodeling of the cancer cellproteome associated with increases in protein biosynthesis.

Massive upregulation of protein synthesis is an onerous expenditure ofcellular resources, and it remains poorly understood how cancer cellsadapt to this increased metabolic load. One example is an increase intotal proteins being synthesized, because cancer cells need to sustainaugmented growth and division. For instance, more than 65% of the energyin the cell is devoted to the bioenergetically expensive process ofprotein synthesis that is greatly increased in most cancers.

Left unchecked, infinite increases in the cancer cell's biosyntheticdemand would tilt the balance from continuous growth and division tocell death. Therefore, increased rates of biosynthetic processes place ahigh demand on cancer cells and are a source of constant stress thatmust be carefully regulated by the activation of appropriatecheckpoints, which remain poorly understood. Increased protein synthesisand the flux in the endoplasmic reticulum (ER) create a state ofproteotoxic stress associated with the accumulation of misfoldedproteins. This ER stress activates the unfolded protein response (UPR).The UPR is composed of three signaling arms: ATF6 (activatingtranscription factor 6) with transcriptional activity to promote ERhomeostasis, IRE1 (inositol-requiring enzyme 1) to control splicing ofthe transcription factor XBP1 enhancing ER gene expression, and PERK[PKR (RNA-activated protein kinase)-like ER-associated protein kinase],which promotes downstream phosphorylation of eIF2α (eukaryoticinitiation factor 2-α) (P-eIF2α) on serine 51. Unlike the other arms ofthe UPR, PERK P-eIF2α creates a direct “brake” for general proteinsynthesis because of the conversion of eIF2α from a substrate of theternary complex, which is necessary to promote the initiation step ofmRNA translation, to an inhibitor of this complex.

Although UPR activation has previously been associated with cancer, itremains poorly understood which oncogenes and/or combinations ofoncogenes control distinct arms of this pathway in vivo during theinitiation or progression of tumor development. It is also unclearwhether and when the UPR is activated during the course of cancerevolution, or its specific requirements in distinct phases oftumorigenesis. Accordingly, there is a need in the art for anunderstanding of how cancer cells accommodate the overwhelming stressassociated with a very high protein synthesis burden. There is a need inthe art for an understanding of the cytoprotective responses activatedin aggressive neoplasms. There is a need in the art for identifyingpoints of vulnerability in cancer's adaptations to massive growth ratesthat can be exploited for therapeutic interventions. Additionally, thereremains a need in the art for elucidation of the pathways by which thecombination of PTEN loss and Myc activation work in concert to drivecancer.

SUMMARY OF THE INVENTION

The inventions disclosed herein are based on the unexpected discoverythat combined PTEN loss and Myc activation results in attenuated proteinsynthesis rates. This observation reveals an interesting paradox,wherein, despite the presence of two oncogenic lesions that individuallyup-regulate protein synthesis, a previously unknown adaptive response isactivated by the combined mutations, and this adaptive brake on proteinsynthesis provides a pro-survival means for aggressive tumors to endurethe deleterious effects of high growth rates.

The inventors of the present disclosure have further discovered that theadaptive response is based upon the selective upregulation of thePERK-mediated integrated stress response (ISR) branch of the unfoldedprotein response (UPR). Thus, PERK activation is a distinct adaptiveresponse that promotes tumorigenesis in aggressive cancer, driven by theunexpected cooperation of two oncogenic lesions.

Furthermore, as demonstrated in the disclosures of the presentapplication, interventions that inhibit the adaptive response willpromote the death of aggressively growing tumor cells and provide anovel target for the treatment of cancer.

Accordingly, in a first aspect, the scope of the invention encompassesnovel methods of treating cancer by inhibiting adaptive responses thatenable aggressive tumors to survive the stress of highly acceleratedgrowth. The scope of the invention encompasses interventions thatinhibit the PERK-eIF2α pathway, which, as shown herein, is specificallyand selectively activated in cells having PTEN loss and activated Myc.

In another aspect, the scope of the invention encompasses novel methodsof prognosing cancer by assessing PTEN loss and Myc activation status.As shown herein, the combination of these factors results in thepromotion of tumor growth by attenuating apoptotic and other stressesthat arise from enhanced protein synthesis. Cancerous cells having thisphenotype are better adapted for aggressive growth and thus thephenotype provides a prognostic measure.

In yet another aspect, the scope of the invention encompasses novelmethods of treating cancer, comprising an assessment of PTEN loss andMyc activation status in the cancerous cells of a subject, and theselection and administration of an appropriate treatment based thereon.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, 1E, and 1F. FIG. 1A depicts total dehydratedprostate weights from 6-week-old mice and FIG. 1B depicts totaldehydrated prostate weights from 10-week-old mice, averaged for eachgenotype (n=3 to 6 mice per arm, mean±SEM). wild-type, WT. FIG. 1Cdepicts phenotypical penetrance percentages for low-grade prostaticintraepithelial neoplasia (LgPIN), HgPIN, and cancer in anteriorprostate tissues from 6- and 10-week-old mice evaluated by hematoxylinand eosin (H&E) staining. FIG. 1D depicts quantification of prostatetumor size in mice with an average age of 8 months (n=5 mice per arm,mean±SEM). FIG. 1F depicts newly synthesized proteins measured by ³⁵Smethionine/cysteine incorporation in organoids relative to WTlittermates (n=5, mean±SEM). *P<0.05, **P<0.01, ***P<0.001, t test.

FIG. 2. FIG. 2 depicts P-PERK expression and P-eIF2α expression,quantified relative to DAPI (n=3 mice per arm, with four images averagedper mouse, mean±SEM).

FIGS. 3A and 3B. FIG. 3A depicts quantification of radioactive pulserelative to loading, depicted as percent over WT (n=3, mean±SEM). FIG.3A depicts quantification of tumor size as fold change relative tobaseline volume at 3- and 6-week time points (mean±SEM). **P<0.01,***P<0.001, t test.

FIGS. 4A and 4B. FIG. 4A depicts quantification of annexin V-positivecells analyzed by flow cytometry relative to control cells aftertreatment with DMSO or 500 nM ISRIB for 9 hours (n=3, mean±SEM) *P<0.05,t test. FIG. 4B depicts Cox proportional hazards regression results in aForest plot of hazard ratios and 95% CI for factors associated with riskof clinical progression after surgery. Independent factors are tumorwith PTEN loss/low P-eIF2α or PTEN loss/high P-eIF2α versus a referencegroup with normal PTEN expression; age in years; PSA in nanograms permilliliter; Gleason score >7 versus 6; and pathological stage T3-T4versus T2 at the time of prostatectomy.

FIGS. 5A, 5B, 5C, 5D, 5E, 5F, and 5G. FIGS. 5A, 5B, and 5C depictquantification of protein expression as relative mean IF intensitynormalized to adjacent stromal tissue for: FIG. 5A: benign, FIG. 5B PCa,and mPCa cell types. FIG. 5D and FIG. 5E depict Kaplan-Meier tumorsurvival curves for mice bearing pPCa (5D) or mPCa (5E) tumors treatedwith ISRIB (10 mg/kg) or vehicle (n=8, per cohort; **P=0.01, log-ranktest). The survival curves represent mice euthanized when tumors reachedan end point of 2 cm or when the mice showed clear signs of morbidity.FIG. 5F depicts quantification of PDX tumors treated with vehicle orISRIB (10 mg/kg); (n=3, ***P<0.001, t test). FIG. 5G depictsquantification of newly synthesized proteins in vivo, assessed byincorporation of OP-Puro within PDX treated with ISRIB (10 mg/kg) orvehicle (n=3 to 4 per arm, mean±SEM; *P<0.05, t test). n.s., notsignificant. MFI, mean fluorescence intensity.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to various aspects of cancer prognosis andtreatment in a subject. The subject may comprise a human patient, or maycomprise a non-human animal, for example a test animal or veterinarysubject. The scope of the invention also extends to the treatment ofcultured cancer cells, cancer explants, or other in vitro uses. Thesubject may comprise a subject at risk of cancer, a subject at risk ofcancer recurrence, a subject having one or more tumors, or a subjectthat has previously had tumors or other cancerous cells removed from thebody, for example, by surgical resection, or has had cancerous cellsablated by a treatment such as radiation therapy, chemotherapy, orimmunotherapy.

In various implementations, the subject has or has had cancer, i.e. hasor has had cancerous cells in the body, for example, one or more tumors.Cancerous cells, as used herein, may be precancerous cells, tumor cells,metastasizing cells, or other cells indicative of cancer risk or cancer.The cancerous cells may be those of any neoplastic condition known inthe art, for example, a cancer selected from the group consisting ofbladder cancer, brain cancer, breast cancer, cervical cancer, colorectalcancer, esophageal cancer, head and neck cancer, kidney cancer, lungcancer, leukemia, lymphoma, myeloma, ovarian cancer, pancreatic cancer,sarcoma, and skin cancer. In one embodiment, the cancer is prostatecancer. For example, the subject may comprise a subject having prostatecancer, at risk of prostate cancer, or a post-surgical subject at riskof prostate cancer recurrence. In some embodiments, the cancerous cellsare leukemia cells, for example, T-cell acute lymphoblastic leukemiacells. In some implementations, the cancer comprises a primary tumor. Insome implementations, the cancer comprises a metastasis.

In some implementations, the methods of the invention may be applied forthe treatment of cancer. “Treatment,” as used herein, comprises anypreventative or therapeutic treatment of cancer, including, for example:prevention of cancer; slowing the progression of cancer; preventing therecurrence of cancer; preventing metastasis; reducing tumor size;slowing tumor growth, and increasing survival time.

Certain methods described herein are accomplished by the administrationof a pharmaceutically effective amount of an agent. A pharmaceuticallyeffective amount, as used herein, means for example, an amountsufficient to cause a measurable biological response, an amountsufficient to cause measurable disruption of adaptive ISR responses, oran amount sufficient to cause any measurable therapeutic effect.

The agents of the invention may be administered at any safe andefficacious dosage, determined as known in the art for the selectedagent. Exemplary dosages may be, for example, in the range of 1 ng agentto 1,000 milligram of agent, depending on the potency, ADMET, and sideeffects of the selected agent. Exemplary dosages are in the range of1-100 ng, 100-1,0000 ng, 1 to 100 micrograms, 100-500 micrograms,500-1,000 micrograms, 1 to 100 mg, 100-500 mg, or 100-1,000 mg. Dosagesmay be administered, for example, daily, multiple times per week,weekly, monthly, or as otherwise effective for the selected agent.

The agents of the invention may be administered in any manner compatiblewith their physical and pharmacological properties. Administration maybe systemic or localized. For example, administration may be orally,intravenously, topically, by intraperitoneal injection, or byintratumoral injection.

The agents of the invention may be administered in combination with anypharmaceutically acceptable carrier, excipient, or delivery vehicle. Theagent may be co-administered in combination with any other therapeuticcomposition, for example an immunotherapy or chemotherapeutic agent.

PTEN Loss and Myc Activation.

In a primary implementation of the invention, the methods of theinvention are applied to a subject wherein cancerous cells of thesubject have a combination of both: (1) PTEN loss and (2) Mycactivation. Accordingly, certain aspects of the invention encompass anassessment of PTEN loss in cancerous cells. PTEN means the phosphataseand tensin homolog, as known in the art. PTEN loss, as used herein,means any reduction or absence of PTEN expression in a cancerous cell.PTEN loss, may include, for example, any reduction in PTEN expression,any reduction in PTEN protein abundance or activity, or any loss (e.g.deletion) of the PTEN gene or the downregulation of the PTEN gene.“Reduction,” in the context of PTEN loss means an observed reductioncompared to a selected baseline tissue. For example, in one embodiment,the assessment of PTEN loss in cancerous cells is made by comparing ameasure of PTEN expression in the cancerous cells against the selectedmeasure of PTEN expression in non-cancerous tissue, for example, fromthe vicinity of the cancerous cells. Any relevant benign stromal cellcan be utilized to establish the baseline. For example, the comparisonof PTEN expression may be made by use of cancerous cells and adjacent orintermixed non-cancerous cells in a sample, for example, a samplecomprising a biopsy or resected tumor. For example, in the case of aresected tumor, non-cancerous cells in the surgically removed margin maybe used as the source of non-cancerous cells for comparison of PTENexpression. In one embodiment, the comparison is made against averagePTEN expression levels in benign cells of like subjects (for examplefrom matched patient pools, e.g., matched by age, demographic factors,disease factors, or other relevant matching factors). For example, PTENexpression of cancerous cells from a prostate tumor may be comparedagainst average PTEN expression in benign prostate tissue observed in apopulation of like subjects.

PTEN loss may be any loss or reduction compared to the selectedbaseline, for example, a reduction in PTEN expression of at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or at least 95%reduced, compared PTEN expression or abundance of the selected baseline.Statistically significant cutoff or threshold values for determinationof PTEN loss may be established, by methods known in the art.

Certain aspects of the invention encompass an assessment of Mycactivation. Myc activation, as used herein, may encompass anysubstantial or elevated expression of the c-myc gene and the presence oractivity of its expression protein, the c-myc transcription factor.Activated Myc may encompass upregulated c-Myc expression, orconstitutive c-Myc expression, for example, by the action of mutationsin the Myc promotor or by the action of upstream species which regulateMyc expression, for example, by the action of upstream species whichregulate the translation of MYC mRNA, MYC degradation or MYC mRNAstability. As with PTEN loss, Myc activation in cancerous cells may beestablished by comparison to a selected baseline. In one embodiment, thebaseline is Myc expression in adjacent, benign non-cancerous cells. Inone embodiment, the baseline is population average Myc expression inbenign tissues of the same type as the cancerous cells (e.g. comparingMyc expression in cells of a prostate tumor to population average Mycexpression in benign prostate tissues).

Designation of Myc activation may be defined by exceeding a selectedthreshold for a selected measure of Myc activation compared to theselected baseline, for example, an increase in Myc activation of atleast 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95% greater than the selected baseline value.

As described below, elevated phosphorylated eIF2α levels may serve as apredictive factor equivalent to the Myc oncogene for prognosing cancerwhen used in combination with PTEN loss. Accordingly, certain aspects ofthe invention are directed to assessment of elevated phosphorylatedeIF2α. Elevated phosphorylated eIF2α activation means any increase in aselected measure of phosphorylated eIF2α abundance, for example,absolute abundance or the ratio of phosphorylated eIF2α tonon-phosphorylated eIF2α. “Elevated” may be any increase, for example,an increase of at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 95% greater than the selected baseline value.

Treatment by Inhibition of Adaptive ISR.

As disclosed herein, the combination of PTEN loss and Myc activationimparts a survival advantage to cancerous cells. Specifically, theinventors of the present disclosure have determined that PTEN loss andMyc activation, in combination, promote an adaptive response to theonerous stress of highly upregulated protein synthesis by theupregulation of the adaptive mechanisms.

In the broadest implementation, the scope of the invention encompasses amethod of treating cancer by inhibiting ISR processes in cancerous cellsthat enable the cell to escape growth stress. The scope of the inventionthus extends to any intervention which disrupts a prosurvival ISRresponse. The ISR response inhibited in the methods of the invention maybe, for example, an IRE1-mediated response, a heme-regulated eIF2α(HRI), general control non-depressible 2 (GCN2) and double stranded RNAdependent protein kinase (PKR)

In a primary embodiment, the methods of the invention are directed toinhibiting the PERK-phosphorylated-eIF2α-mediated integrated stressresponse. The inventors of the present disclosure have determined thatthis axis of the UPR is selectively triggered in tumor cells lackingPTEN and having activated Myc and that this results in activation of thePERK-mediated branch of the integrated stress response (ISR) While otherbranches of the UPR may be upregulated generally in response to tumors,the PERK-P-eIF2α ISR branch of the UPR is shown herein to bespecifically and selectively activated in cells having PTEN loss andactivated Myc.

Activated Myc results in elevated eIF2-P, the phosphorylated form ofeukaryotic initiation factor (eIF2). This species is a key regulatoryfactor in the initiation of eukaryotic translation. In theunphosphorylated form, eIF2 (itself a heterodimer), forms a quaternarycomplex with other factors that may continue on to affect thetranslation of mRNAs. The formation of this complex is inhibited,however, when eIF2 is phosphorylated. eIF2 has a phosphorylation site onits alpha subunit, which may be acted on by any number of kinases whichregulate translation. eIF2 may be phosphorylated by activated PERK(protein kinase RNA-like endoplasmic reticulum kinase), a regulatoryelement which itself is activated by ER stress.

In a first and general aspect, the scope of the invention encompasses amethod of treating cancer by any process which inhibits the ability ofPTEN- and Myc activated cells to adapt to the UPR stress induced by highlevels of protein synthesis.

In a primary aspect, the scope of the invention encompasses thetreatment of cancer in subjects having (or having had) cancerous cells,wherein the cancerous cells have both (1) PTEN loss and (2) activatedMyc, by inhibition of the PERK-mediated ISR. Such inhibition may includeany intervention that disrupts the activation, magnitude, or efficiencyof adaptive response mediated by the PERK-eIF2 pathway.

The inhibition of the adaptive response may be achieved by variousmeans, including by the administration of an agent comprising aninhibitor of the PERK-mediated ISR. As used herein, an “inhibitor of thePERK-mediated ISR” is an agent wherein the administration of the agentresults in one or more effects such as: inhibition of phosphorylation ofeIF2α by PERK;

-   -   depletion or inactivation of phosphorylated eIF2α;    -   an increase in the relative abundance of unphosphorylated eIF2α        to phosphorylated eIF2α;    -   rendering cells insensitive to the effects of phosphorylated        eIF2α;    -   promotion of the formation of the quaternary complex;    -   increase in the rate of protein synthesis in the cancerous        cells; and    -   increased levels of cancerous cell death caused by protein        synthesis stress; and    -   any other inhibition of the activation of the adaptive brake on        global protein synthesis impacted by the PERK-mediated ISR.

In one embodiment, the inhibitor of the PERK-mediated ISR is an agentwhich renders cells insensitive to the effects of phosphorylated eIF2α.For example, In one embodiment, the agent which renders cellsinsensitive to the effects of eIF2α phosphorylation comprises ISRIB(“Integrated Stress Response inhibitor”), which is a potent andselective PERK inhibitor with IC50 of 5 nM. ISRIB increases the activityof the eIF2B guanosine recycling factor by stabilizing it into a decamerholoenzyme that enhances the binding of the eIF2 factor, therebyrestoring protein synthesis regardless of eIF2α phosphorylation levels.The scope of the invention extends to the use of ISRIB variants andderivatives, for example, derivatives as described in U.S. Pat. No.9,708,247, entitled “Modulators of the eIF2alpha pathway,” by Walter etal.

In one embodiment, the PERK-eIF2 inhibitor comprises an inhibitor ofPERK kinase activity. Exemplary inhibitors of PERK kinase activityinclude GSK2606414 and GSK2656157, and LY-4, as known in the art. In oneembodiment, the PERK inhibitor is a composition described in U.S. Pat.No. 8,598,156, entitled “Chemical Compounds,” by Axten et al. In oneembodiment, the PERK-eIF2 inhibitor comprises a composition described inU.S. Pat. No. 9,668,662, entitledN-(2,3-dihydro-1H-pyrrolo[2,3-b]pyrdin-5-yl)-4-quinazolinamine andN-(2,3-dihydro-1H-indol-5-yl)-4-quinazolinamine derivatives as perkinhibitors, by Stansfield et al. In one embodiment, the inhibitor ofPERK kinase activity comprises a composition described in Atkins et al.,Characterization of a Novel PERK Kinase Inhibitor with Antitumor andAntiangiogenic Activity, 2013 Mar. 15; 73(6):1993-2002.

In some implementations of the invention, the inhibitor of PERK-mediatedISR comprises a composition which disrupts the adaptive response at thegenetic level by targeting the expression of genes involved in theadaptive response. The inhibitor may comprise a species which disrupts atarget gene, wherein the target gene comprises PERK, eIF2, ATF4 or anyother element of the adaptive response. Exemplary compositions includeTALENs, siRNA vectors, and CRISPR-Cas9 or like constructs directed tothe selected target gene.

In some implementations, the inhibitor of PERK-mediated ISR comprises acomposition which disrupts the adaptive response at the protein level bytargeting the proteins involved in the adaptive response. The inhibitormay comprise a species which disrupts a target protein, wherein thetarget protein comprises PERK, eIF2, ATF4, or other elements of theadaptive response. Exemplary compositions include PROTEK or otherselective ubiquitination complexes that selectively promote thedegradation of PERK or eIF2-P. Agents may also comprise agents that bindto and inactivate or block access to active sites of target proteins.Exemplary species include aptamers, antibodies, and dominant negativemutants.

ATF4 Intervention.

The inventors of the present disclosure have further determined thatactivation of P-PERK and P-eIF2α results in the activation of ATF4, aknown target of the PERK-P-eIF2α axis. ATF4 is selectively translatedduring eIF2α-induced global inhibition of protein synthesis and is atranscription factor that regulates a wide range of pro-adaptive geneswhich enable adaptation to stress conditions. The inventors of thepresent disclosure have demonstrated herein that ablation of ATF4activity will result in tumor death. Accordingly, in one aspect, thescope of the invention encompasses treatments which inhibit (i.e. reducethe expression or activity) of ATF4. In one aspect, the scope of theinvention comprises the administration of an agent comprising aninhibitor of the PERK-mediated ISR comprising an ATF4 inhibitor.

In one embodiment, the ATF4 inhibitor comprises ursolic acid ortomatidine, or a derivative thereof, or a compound disclosed in U.S.Pat. No. 9,034,299, entitled “ATF4 inhibitors and their use for neuralprotection, repair, regeneration, and plasticity,” by Ratan; UnitedStates Patent Application Publication Number 20160317526, entitled,“Prolylhydroxylase/atf4 inhibitors and methods of use for treatingneural cell injury or death and conditions resulting therefrom,” byRatan and Karuppagounder; and PCT International Patent ApplicationNumber WO2017212423, entitled “Chemical Compounds,” by Axten et al.

Prognostic and Diagnostic Methods.

As above, the inventors of the present disclosure have determined thatPTEN loss and Myc activation imparts a survival advantage byupregulation of the adaptive ISR. For example, as shown herein, in thecase of prostate cancer, assessment of PTEN loss and activated Mycenables discrimination of individuals that will experience metastaticprogression or prostate cancer-specific mortality. Accordingly, thepresent disclosures provide the art with a simplified, two-factor panelfor prognosing various aspects of cancer pathology and progression.

Accordingly, the scope of the invention encompasses a method ofdetermining a diagnostic status in a subject, comprising the steps of

-   -   obtaining a sample from the subject, wherein the sample        comprises cancerous cells;    -   assessing PTEN loss and Myc activation in the cancerous cells;        and    -   if both PTEN loss and Myc activation are present, assigning an        associated diagnostic status to the subject.        The diagnostic status may be any diagnostic or prognostic status        associated with the combination of PTEN loss and Myc activation.        For example, the diagnostic status may be any one or more of:    -   amenability to treatment by inhibition of adaptive ISR;    -   increased likelihood of cancer progression;    -   increased likelihood of cancer recurrence;    -   increased likelihood of cancer metastasis;    -   increased likelihood of cancer mortality or decreased survival        time.

The sample may comprise any relevant sample containing cancerous cells,including tumor biopsy, such as a punch biopsy, fine needle aspirationbiopsy, needle core biopsy, bone biopsy or surgical specimen. Thecancerous cells may comprise tumor cells, for example, primary tumorcells, cells from metastasis, etc. and precancerous cells, for example,putatively precancerous cells wherein tumorigenesis is not fullyestablished.

The assessment of PTEN loss and Myc activation may be made bymeasurement of PTEN and Myc. Alternatively, phosphorylated eIF2α may beused as a measure of Myc activation, wherein elevated phosphorylatedeIF2α is indicative of Myc activation.

Assessment of biomarker abundance may be performed by any appropriatemethod. In one implementation, expression levels or abundance aredetermined by direct measurement of expression at the protein or mRNAlevel, for example by microarray analysis, quantitative PCR analysis, orRNA sequencing analysis. Alternatively, labeled antibody systems may beused to quantify target protein abundance in the cells, followed byimmunofluorescence analysis, such as FISH analysis.

In the case of PTEN, PTEN expression or PTEN protein levels may beassayed by appropriate methods known in the art. For example, PTEN maybe quantified as described in United States Patent ApplicationPublication Number 20100303809, entitled “Methods for the Detection andQuantitation of PTEN,” by Bacus and Sakr et al., 2010, “Protocol forPTEN Expression by Immunohistochemistry in Formalin-fixedParaffin-embedded Human Breast Carcinoma,” Appl Immunohistochem MolMorphol. 2010 July; 18(4): 371-374. PTEN expression can be assayed viaimmunohistochemistry (IHC) or immunofluorescence (IF) analysis and isquantifiable by mean fluorescence intensity, for example by comparingthe differential expression of PTEN in stromal benign tissue to that ofthe cancerous cells.

In one implementation, eIF2α-P, the phosphorylated form of eIF2, ismeasured directly by immunohistochemistry or immunofluorescence (IF) forexample, by using antibodies against phosphorylated forms of eIF2αprotein. eIF2α-P may be assessed by any means known in the art, forexample, as described in Teske et al., 2011, Methods in Enzymology,Chapter Nineteen—Methods for Analyzing eIF2 Kinases and TranslationalControl in the Unfolded Protein Response, Volume 490, pp 333-356 andLobo et al., 2000, “Levels, Phosphorylation Status and CellularLocalization of Translational Factor EIF2 in GastrointestinalCarcinomas,” Histochemical Journal pp 139-150. Abundance may bequantified by mean fluorescence intensity, for example, by comparing thedifferential abundance of eIF2α in benign stroma and cancerous tissue.

In some embodiments, elevated eIF2-P may be assessed by measurement ofMyc activation, for example by measurement of Myc gene expression orprotein levels, wherein elevated Myc expression is indicative ofactivated elevated eIF2α-P. For example, Myc may be detected andquantified as described in Hilpert et al., 2001, “Anti-c-myc antibody9E10: epitope key positions and variability characterized using peptidespot synthesis on cellulose,” Protein Engineering, Design and Selection,Volume 14, Pages 803-806 and U.S. Pat. No. 4,918,162, “Assays andantibodies for N-MYC proteins,” by Slalom. MYC protein level can beobtained by IF analysis and is quantifiable by mean fluorescenceintensity by comparing the differential expression in benign stroma andcancerous tissue.

If the cells in the sample are determined to have both PTEN loss andelevated activated Myc, an associated diagnostic status may be assignedto the subject. Exemplary diagnostic status associated with PTEN lossand Myc activation include elevated risk for the development of cancer;elevated risk of cancer relapse; elevated risk of aggressive cancer;elevated risk of metastasis; elevated risk of cancer progression; or anincreased likelihood that cancer that will respond to inhibitors ofadaptive ISR. The risk factors may be assessed for subjects pre- orpost-treatment, for example, In one embodiment, the factor is elevatedrisk of cancer recurrence progression in subjects having previouslyreceived curative treatment, such as surgery, radiation, chemotherapy,and/or immunotherapy.

The precise relationship between PTEN loss/activated Myc status and theselected diagnostic status may be established by any statistical methodor model known in the art. For example, a classifier or other predictivemodel may be applied to the measured levels of PTEN and Myc or eIF2-P,for example, a classifier or predictive model generated usingstatistical methods such as: machine learning classifiers such as randomforest, support vector machines, and newer deep learning and neuralnetwork approach and other statistical model generating methods known inthe art. The output of the model may be a classification, score, orother output indicative of the subject's risk or probability of havingthe selected diagnostic status.

Upon determination that the cancerous cells of a subject have the PTENloss/activated Myc phenotype, a suitable treatment may be administered.In one embodiment, the suitable treatment is the administration of aninhibitor of adaptive ISR. In one embodiment, the suitable treatment isa more aggressive treatment than would be administered to subjects withcancerous cells lacking the PTEN loss/activated Myc phenotype, forexample, more aggressive surgical interventions, chemotherapeuticinterventions, radiological interventions, or immunotherapyinterventions, in line with the increased risk associated with theprosurvival phenotype.

Diagnostic Kits.

In one aspect, the scope of the invention comprises a diagnostic kit forthe assessment of the PTEN loss and activated Myc phenotype. Thediagnostic kit will comprise a collection of two or more compositions ofmatter directed to the detection and/or quantification of PTEN and Mycor eIF2-P in a sample. The two or more components may be packaged in acommon packaging element, for example, a box, packet, or other commoncontainer. For example, the kit may comprise fluorescently labeledantibodies, or antibodies otherwise configured for immunofluorescentdetection to PTEN and Myc or eIF2α-P, PCR primers for the amplificationof PTEN and Myc or eIF2α in a sample, or other reagents for theselective amplification, labeling, or detection of PTEN and Myc oreIF2α-P. The kit may further comprise components for detection and/orquantification of PTEN and Myc or eIF2-P in a sample, for example,buffers, sample collection and analysis vessels, and software foranalyzing images.

In one embodiment, the diagnostic kit comprises an antibody for thedetection of PTEN and one of (1) an antibody for the detection of Myc or(2) an antibody for the detection of phosphorylated eIF2α.

EXAMPLES Example 1. MYC Amplification with PTEN Loss Diminish OncogenicIncreases of Global Protein Synthesis in Lethal Murine PCa

Distinct stages of human PCa were modeled in the mouse, using a newlygenerated conditional transgenic MYC mouse, where the overexpression ofC-MYC is driven in a Cre-specific manner (Myc^(Tg)), in combination withthe conditional loss of PTEN in the prostate epithelium (Pb-cre4;Pten^(fl/fl), herein referred to as Pten^(fl/fl)), as described inLesche R, Groszer M, Gao J, Wang Y, Messing A, Sun H, Liu X, Wu H.Cre/loxP-mediated inactivation of the murine Pten tumor suppressor gene.Genesis. 2002; 32:148-149. [PubMed: 11857804]. The advantage of thismouse is that cells overexpressing Myc^(Tg) can be traced throughexpression of green fluorescent protein (GFP) present in the targetinglocus, allowing for visualization of the earliest events intumorigenesis. In agreement with the notion that MYC hyperactivation maybe a secondary event for human PCa development, it was observed that MYCoverexpression alone in prostate epithelium (Pb-cre4; Myc^(Tg), hereinreferred to as Myc^(Tg)) increased proliferation but did not result inadenocarcinoma by 1 year of age. This is consistent with previousreports, which showed MYC expression under the control of similarpromoters to those used here. Myc^(Tg) mice with concomitant loss ofPTEN in prostate tissue (Pten^(fl/fl); Myc^(Tg)) showed significantenlargement of prostate growth by 6 weeks of age (P<0.0003) andaccelerated development of high-grade prostatic intraepithelialneoplasia (HgPIN) compared to mice with loss of PTEN alone. PTEN lossand MYC amplification cooperated to develop adenocarcinoma by 10 weeks(FIG. 1B), resulting in marked increases in Pten^(fl/fl); Myc^(Tg) tumorsize visualized by ultrasound (FIG. 1C). This aggressive oncogenicprogression significantly decreased overall survival (P<0.05), with amean life span of 75 weeks (FIG. 1D). Collectively, this geneticallyengineered mouse model (GEMM) recapitulates aggressive human PCa andresults in decreased survival.

To evaluate the effects of these key oncogenes on global proteinsynthesis, newly synthesized proteins were assessed by incorporation of³⁵S-labeled methionine in organoid cultures. Primary mousethree-dimensional organoid cultures were established to recapitulate thecellular environment of the murine prostate gland ex vivo. Organoidswere derived from dissociated mouse prostate tissue containing a mixedpopulation of luminal and basal cell types to mimic the histologyobserved in vivo. Western blot analysis confirmed that Myc^(Tg)expression and PTEN loss were evident and associated with increased GFPexpression and AKT phosphorylation. Consistent with the known ability ofthese major oncogenic pathways to increase protein synthesis, eitherloss of PTEN or MYC hyperactivation significantly increased globalprotein synthesis by about 20% (P<0.0003 for both). On the contrary, itwas observed that an unanticipated but significant dampening in globalprotein synthesis occurred in Ptenfl/fl; MycTg mice (P=0.01), despitethe fact that these mice developed more aggressive PCa (FIG. 1D). Thisobservation revealed an interesting paradox. It suggested that despitethe presence of two oncogenic lesions that individually up-regulateprotein synthesis, an adaptive response appears activated when proteinsynthesis is up-regulated beyond a specific threshold in aggressive PCa.

Example 2. Aggressive PCa Activates a Key Cellular Stress ResponseDuring Tumor Development

Proteins that are synthesized in the secretory pathway amount to about30% of the total proteome in most eukaryotic cells. Although UPRactivation can be studied with pharmacological inducers of ER stress,under physiological processes, the activation of the UPR may reduce theunfolded protein load through several prosurvival mechanisms, includingthe expansion of the ER membrane and the selective synthesis of keycomponents of the protein folding and quality control machinery. Toaddress how cancer cells respond and adapt to a protein synthesis burdenin vivo and downstream of specific oncogenic lesions, it was testedwhether a specific molecular signature of the UPR may be activated inPten^(fl/fl)-versus Pten^(fl/fl); Myc^(Tg)-derived PCa.

Quantitative immunofluorescence (IF) staining was performed for cleavedATF6, P-IRE1, and P-PERK during tumor development to test whether theUPR was activated during PCa progression. Visualizing UPR expressionwithin prostatic tissue at 10 weeks of age allowed direct assessment ofthe activity of each arm during neoplasia. Whereas the ATF6 and IRE1branches of the UPR were relatively equally activated in Pten^(fl/fl)and Pten^(fl/fl); Myc^(Tg) tissue, PERK phosphorylation was selectivelyincreased by over 15-fold within Pten^(fl/fl); Myc^(Tg) tissue comparedto its near absence in Pten^(fl/fl) cells (FIG. 2). Thus, PERKactivation is a distinct response that may promote tumorigenesis inaggressive PCa driven by the cooperation of two oncogenic lesions. Toconfirm the selective activation of PERK signaling in Pten^(fl/fl);Myc^(Tg) mice, the downstream signaling to eIF2α was evaluated. P-eIF2αwas also markedly increased in Pten^(fl/fl); Myc^(Tg) mice and strongestwithin areas of PIN but remained absent within Pten^(fl/fl) tissues(FIG. 2). The expression of the ER-specific molecular chaperone BiP wasnot changed and was also high in normal prostatic tissues in agreementwith the secretory role of these glands. Collectively, this analysisreveals two independent, yet linked mechanisms: (i) activation of eachUPR pathway in PCa in vivo and (ii) activation of a P-eIF2α-dependentresponse selectively in Pten^(fl/fl); Myc^(Tg) mice, which display moreaggressive PCa progression and reduced survival.

Example 3. Rebalancing Protein Synthesis Through P-eIF2α is Required forAggressive PCa Progression

A general UPR response may promote adaptation to proteotoxic and ERstress, whereas the activation of P-eIF2α could place a direct brake onthe overwhelming burden of protein synthesis that occurs during moreaggressive tumorigenesis. To test this hypothesis, the organoid cultureswere employed, which recapitulate the in vivo phenotype. ThePten^(fl/fl); Myc^(Tg) cultures showed increased activation of P-PERK,P-eIF2α, and expression of ATF4, which is a known target of thePERK-P-eIF2α axis (FIG. 3A). To determine whether the activation of thisadaptive response was altering global protein synthesis, asmall-molecule inhibitor of P-eIF2α activity, ISRIB was used, a compoundthat selectively reverses the effects of eIF2α phosphorylation.Specifically, P-eIF2α binds its dedicated guanine nucleotide exchangingfactor (GEF), eIF2B, with enhanced affinity relative to eIF2α. Thus,P-eIF2α sequesters eIF2B from interacting with eIF2α to exchangeguanosine diphosphate with guanosine triphosphate, which is an essentialstep to form the translation preinitiation complex. ISRIB increaseseIF2B GEF activity by stabilizing it into a decamer holoenzyme toenhance the binding of the eIF2 factor, thereby restoring proteinsynthesis regardless of eIF2α phosphorylation. In Pten^(fl/fl) organoidcultures, protein synthesis was not altered by ISRIB treatment, despitethe drug inhibiting P-eIF2α activity, as confirmed by a decrease in ATF4expression (FIG. 3A). Conversely, a marked increase of newly synthesizedproteins was observed in Pten^(fl/fl); Myc^(Tg) cells, which showincreased P-eIF2α signaling (FIG. 3A). Together, these experimentsindicate that P-eIF2α creates an adaptive response to relieve the burdenof increased protein synthesis within Pten^(fl/fl); Myc^(Tg) oncogeniccells.

In addition to PERK, other kinases can phosphorylate the eIF2α subunitupon distinct stress signals: GCN2 (amino acid deprivation), PKR (viralinfection), and HRI (heme deprivation) (30). To assess whether theselective adaptive response observed during aggressive PCa developmentof Pten^(fl/fl); Myc^(Tg) mice was specific to the PERK-P-eIF2α axis, agenetic approach was employed, using Perk^(fl/fl) mice to evaluate theloss of PERK in the prostate gland. Pten^(fl/fl); Myc^(Tg); Perk^(fl/fl)mice showed markedly reduced prostate growth compared to Pten^(fl/fl);Myc^(Tg) mice, with weights similar to Pten^(fl/fl) and Pten^(fl/fl);Perk^(fl/fl) mice at 10 weeks of age. The reduction in prostate sizecorresponded to a decrease in cancer progression and in cellproliferation. To determine the consequence of PERK loss for P-eIF2αsignaling in PCa development, P-eIF2α expression was monitored by IFstaining. The activation of P-eIF2α was reduced by 70% in Pten^(fl/fl);Myc^(Tg); Perk^(fl/fl) tissue compared to Pten^(fl/fl); Myc^(Tg). Thesedata strongly suggest that the P-eIF2α-dependent adaptive stressresponse is driven to a large extent by PERK signaling.

These studies demonstrated that P-eIF2α is directly activated in theearly stage of Pten^(fl/fl); Myc^(Tg) tumorigenesis, being visible inbenign tissue and increasing in HgPIN, which may reflect a distinctpoint of vulnerability for aggressive PCa. To evaluate the necessity ofP-eIF2α for promoting tumor growth or maintenance in vivo, a preclinicaltrial was conducted. Mice with developed tumors were imaged by magneticresonance imaging (MRI) to confirm a measurable baseline of prostatevolume per mouse and then grouped into cohorts for either vehicle orISRIB treatment daily over the course of 6 weeks. Pten^(fl/fl); Myc^(Tg)mice showed tumor regression within 3 weeks of ISRIB treatment, with nosigns of toxicity, whereas all Pten^(fl/fl) mice showed continued tumorgrowth (FIG. 3B).

By 6 weeks, Pten^(fl/fl) mice showed an approximate 40% increase ingrowth over individual baseline measurements, whereas ISRIB-treatedPten^(fl/fl); Myc^(Tg) mice demonstrated no progression in tumor size.In addition, the immune cell infiltration was evaluated, marked by thepan-leukocyte antibody CD45 after 3 weeks of ISRIB treatment andobserved no significant changes regardless of prostate tumor genotypeand treatment. Further analysis of immune cell populations did notdemonstrate substantial differences in total T cell or myeloidpopulations, including dendritic cells, macrophages, and neutrophils. Ofthe intertumoral immune cells examined, less than 5% were either CD4⁺ orCD8⁺ T cells, as expected for the Pten^(fl/fl) murine prostate model.Although exclude the possibility that ISRIB may be remodeling tumorimmunity during initial treatment may be considered, this was notevident after 3 weeks of treatment. Together, these studies reveal thatP-eIF2α signaling is functionally relevant in aggressive PCa and thatthis adaptive response is therapeutically targetable in vivo using thesmall-molecule inhibitor ISRIB.

To extend these observations directly to human disease, human cell lineswere created to mimic the genetic mouse models. Human RWPE-1 epithelialcells were created to stably knock down PTEN (shPTEN) with or withoutMYC overexpression. The combination of PTEN loss with increased MYCexpression activated PERK signaling and P-eIF2α, showing that theadaptive response that we had observed in mice is also triggered inhuman prostate cells. To understand the requirement for this stressresponse checkpoint in human cells, each cell line was treated withISRIB and observed a marked increase in apoptosis, independent ofalterations in proliferation, specifically in shPTEN; MYC^(OE) cellsrelative to control samples (FIG. 4A).

Example 4. High P-eIF2α Expression with Loss of PTEN is Associated withan Increased Risk of Metastasis after Surgery

To further examine the clinical relevance of high P-eIF2α downstream ofPTEN loss, a human tissue microarray (TMA) consisting of 424 tumors wasmade and analyzed the expression of PTEN, c-MYC, and P-eIF2α. On thebasis of the GEMMs, it was predicted that the combination of PTEN lossand P-eIF2α would associate with advanced PCa. An array of patients wasselected with PCa ranging from low to high risk, who received surgery asa curative treatment with a median of 10 years of follow-up toaccurately evaluate the incidence of clinical progression, a compositeoutcome representing visceral or bone metastasis or PCa-specificmortality (MET/PCSM). Quantitative IF of P-eIF2α, c-MYC, and PTEN wasnormalized to adjacent benign tissue and then associated risk wasevaluated for MET/PCSM. After controlling for age, prostate-specificantigen (PSA), Gleason score, and pathological staging, the analysisshowed that patients with PTEN loss/high MYC expression were more likelyto experience metastatic progression than patients with PTEN loss orhigh MYC alone.

These data demonstrated that P-eIF2α is a targetable adaptive responsedownstream of PTEN loss and MYC hyperactivation. Hence, next examinedwas the associated risk of progression in patients with PTEN loss andhigh P-eIF2α at the time of surgery. The rate of MET/PCSM-free survivalwas significantly lower in patients with high P-eIF2α and PTEN losscompared to PTEN loss alone (P<0.01). Only 4% of patients with PTEN lossand low P-eIF2α succumbed to metastasis or death, whereas 19% ofpatients with PTEN loss and high P-eIF2α showed MET/PCSM by 10 yearsafter surgical intervention with the intention to cure the disease.Furthermore, patients with high P-eIF2α and PTEN loss had a higher riskof MET/PCSM compared to patients with no PTEN loss, with a hazard ratioof 5.40 [95% confidence interval (CI), 2.46 to 11.86; P<0.01], whereasother variables that may affect the risk were not significantlydifferent (FIG. 4B). MYC overexpression with either low or high P-eIF2αdid not associate with increased risk of MET/PCSM, supporting thefindings that MYC alone does not drive PCa. Notably, high P-eIF2αexpression played a role equivalent to the MYC oncogene in combinationwith loss of PTEN at predicting metastatic progression, yet unlike MYC,P-eIF2α may be a druggable target. Together, the combination of P-eIF2αand PTEN loss thus serves as a predictor for cancer progression aftercurative treatment, which is independent of the traditional riskassessment system using PSA, cancer grade, and cancer stage.

Next was evaluated the discriminatory properties of high P-eIF2α andPTEN loss as a prognostic marker independent from the most commonly usedrisk assessment score in the clinic, CAPRA-S(Cancer of the Prostate RiskAssessment after Surgery). The c-index (concordance index) was used toevaluate the ability of the protein signature of high P-eIF2α with lossof PTEN to discriminate between individual patients who did or did notsuccumb to metastasis or death after surgery. Currently, cliniciansdepend on genomic risk to individualize treatment decisions using threeavailable gene expression tests: PROLARIS™ (Myriad Genetics Inc.),DECIPHER™ (GenomeDX Inc), and ONCOTYPEDX™ (Genomic Health, Inc). ThePROLARIS™ test relies on the average expression of 31 cell cycleprogression (CCP) genes and was validated using the same cohort ofpatients used in the TMA. Within the same patients, the PROLARIS™-CCPpanel has a combined c-index of 0.77 (CAPRA-S+CCP) (Cooperberg et al.Validation of a cell-cycle progression gene panel to improve riskstratification in a contemporary prostatectomy cohort. J Clin Oncol.2013; 31:1428-1434. [PubMed: 23460710]), whereas high P-eIF2α and PTENloss has a c-index of 0.80. These findings show that concurrent highP-eIF2α and PTEN loss serves as an independent predictor with improvedprognostic accuracy over standard clinicopathologic testing fordiscriminating which individuals may experience metastatic progression.

Example 5. P-eIF2α is a Targetable Adaptive Response in Aggressive HumanPCa

Next, it was sought to functionally evaluate whether the UPR pathwaycould be targeted, specifically through P-eIF2α, in advanced human PCa.Although it is historically difficult to generate human prostatepatient-derived xenograft (PDX) models, this was successfully achievedwith similar characteristics to the Pten^(fl/fl); Myc^(Tg) mice toassess the effects of ISRIB on cancer growth and mortality. Inparticular, two PDX models were generated: one derived from a primarytumor, herein referred to as pPCa, and one derived from a lymph nodemetastasis in the left internal iliac chain from the same patient,herein referred to as mPCa. The pPCa-PDX tumor had significantly lowerMYC expression than the mPCa-PDX tumor (P<0.01), but both showed loss ofPTEN with increased P-AKT expression. A significant increase in P-eIF2αwas observed only in the mPCa (P<0.01).

To test the therapeutic efficacy of ISRIB in human PCa, a preclinicaltrial was performed on the stably passaged PDX model. Targeting P-eIF2αpharmacologically significantly prolonged survival in mice bearing themetastatic tumor with high P-eIF2α (P<0.01; FIGS. 5D and 5E), whereasthe effectiveness of ISRIB treatment was short-lived in pPCa tumor.Consistent with the GEM model, the mPCa-PDX model, with high expressionof P-eIF2α, displayed significant tumor regression and cell death(P<0.01), as demonstrated by increased terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)staining and cleaved caspase 3 expression after only 9 days of ISRIBtreatment (FIGS. 5, D and E, and FIG. S7B). Conversely, the pPCa-PDXmodel, with low P-eIF2α, did not show regression but stabilized witheventual tumor regrowth and no significant cell death. These findingsdemonstrate that attenuating P-eIF2α activity with ISRIB elicits apotent antitumor effect in a humanized model of advanced PCa.

Next, it was determined whether a metastatic PCa tumor, harboring highMYC and loss of PTEN activity in a complex genetic background of humanPCa, relies on eIF2α phosphorylation as an adaptive response to restrainglobal protein synthesis. Therefore, newly synthesized proteins in vivowere assessed by measuring the incorporation of O-propargyl-puromycin(OP-Puro) within the primary and metastatic tumor-derived PDXs, whichhave low or high P-eIF2α, respectively. Upon ISRIB treatment, a markedincrease was observed in global protein synthesis specifically in themPCa PDX, but no change in pPCa tumors where P-eIF2α expression was notup-regulated (FIG. 5G). To further assess the functional relevance ofP-eIF2α signaling, ATF4 expression was decreased in vivo usingintratumor knockdown by small interfering RNA (siRNA). Within the areaof intratumor ATF4 loss, apoptosis and decreased proliferation assessedby TUNEL and Ki67 staining of mPCa PDX was observed. This demonstratedthat inhibition of the PERK-eIF2α axis by a genetic or pharmacologicalapproach effectively results in cell death of aggressive PCa in vivo.

Example 6. Targeting P-eIF2α Activity Reduced Metastasis and ProlongedSurvival in a PDX Model of Metastatic Castration-Resistant PCa

In hormone-sensitive metastatic PCa, androgen deprivation therapy (ADT)remains the mainstay treatment; however, these tumors inevitably developresistance to ADT and progress into the lethal form of metastaticcastration-resistant PCa (mCRPC). Characterization of thehormone-sensitive metastatic disease has not been predictive of outcomesin the clinical setting of lethal mCRPC. To directly study thecontribution of P-eIF2α to metastasis, an additional PDX (herein mCRPCPDX) was generated derived from a patient with mCRPC despite prolongedtreatment with complete androgen blockage using leuprolide (ADT) andantiandrogen therapy (enzalutamide). Three weeks after implantation ofthe mCRPC tumor under the mouse renal capsule, tumor dissemination tothe liver, distant kidney, lymph nodes, and spleen was observed. ThemCRPC PDX line continued to exhibit metastatic dissemination in themouse host after multiple passages and retained histological andmolecular characteristics of the original tumor. The distant metastaticlesions exhibited loss of PTEN, high MYC, and high P-eIF2α expression.

To examine the role of P-eIF2α from the early stages of metastaticgrowth to late stages of dissemination, a prostate-specific membraneantigen [⁶⁸Ga-PSMA-11 PET/computed tomography (CT)] scan was used totrace the progression of very small metastases from early to late stagesof dissemination, which were not visible by conventional imagingmodalities such as ¹⁸F-DG PET/CT. Prostate-specific membrane antigen(PSMA) is highly expressed on the surface of PCa cells and allowssensitive staging to evaluate therapy response in the clinical setting.Mice bearing liver or distal metastasis (confirmed by PSMA PET) weretreated with either vehicle or ISRIB. Inhibition of P-eIF2α with ISRIBsignificantly prolonged survival in mCRPC PDX mice bearing distalmetastatic lesions (P=0.01). In contrast, mice with metastasis diedwithin 10 days on vehicle treatment. By direct imaging with PSMA PET/CT,substantial metastatic regression was observed at distal sites in micetreated with ISRIB. In addition, a difference in metastatic progressionin the liver was confirmed by pathohistological analysis at time ofeuthanasia. Therefore, two independent PDX models of metastatic disease,one derived from a patient with early nodal metastasis(hormone-sensitive) and the second from a patient withcastration-resistant PCa, demonstrated that blocking the activation ofthe adaptive brake on global protein synthesis via the P-eIF2α axisresulted in profound tumor regression and inhibition of metastaticdissemination.

Example 7. Discussion

The data disclosed herein reveal a cell-autonomous mechanism wherein theactivity of two major oncogenic lesions, loss of PTEN and MYCoverexpression, which independently enhance protein synthesis,paradoxically, decrease global protein production when these oncogenicevents coexist. This highlights the requirement for an adaptive proteinhomeostasis response to sustain aggressive tumor development.

Proteostasis is essential for normal cell health and viability, and assuch is ensured by the coordinated control of protein synthesis,folding, and degradation. Although the UPR enables proteostasis to berestored during unfavorable conditions, herein is demonstrated that PCacells have usurped a specific branch of this pathway for tumor growthand maintenance. The UPR consists of three main branches, yet only thePERK-P-eIF2α axis is selectively triggered in this pathophysiologicalstate to ensure continued survival of cancer cells. The adaptiveresponse involving P-eIF2α signaling provides a barrier to uncontrolledincreases in protein synthesis and creates a permissive environment forcontinued tumor growth.

All patents, patent applications, and publications cited in thisspecification are herein incorporated by reference to the same extent asif each independent patent application, or publication was specificallyand individually indicated to be incorporated by reference. Thedisclosed embodiments are presented for purposes of illustration and notlimitation. While the invention has been described with reference to thedescribed embodiments thereof, it will be appreciated by those of skillin the art that modifications can be made to the structure and elementsof the invention without departing from the spirit and scope of theinvention as a whole.

What is claimed is:
 1. A method of treating cancer in a subject,comprising the steps of: obtaining a sample comprising cancerous cellsfrom the subject; assessing PTEN loss in the cancerous cells; assessingMyc activation in the cancerous cells; and wherein, if both PTEN lossand Myc activation in the cancerous cells is observed, administering tothe subject a pharmaceutically active amount of an agent which inhibitsthe PERK-mediated integrated stress response, wherein the cancerouscells of the subject comprise prostate cancer cells.
 2. The method ofclaim 1, wherein the agent which inhibits the PERK-mediated integratedstress response comprises an agent which renders cells insensitive tothe effects of eIF2α phosphorylation.
 3. The method of claim 2, whereinthe agent which inhibits the PERK-mediated integrated stress responsecomprises ISRIB.
 4. The method of claim 1, wherein the agent whichinhibits the PERK-mediated integrated stress response comprises aninhibitor of PERK expression, translation, or kinase activity.
 5. Themethod of claim 4, wherein the inhibitor of PERK expression,translation, or kinase activity comprises GSK2606414 or GSK2656157. 6.The method of claim 1, wherein the agent which inhibits thePERK-mediated integrated stress response is an ATF4 inhibitor.